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BACKGROUND: Glucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools. METHODOLOGY/PRINCIPAL FINDINGS: MYB28 was identified as a candidate regulator of aliphatic glucosinolates based on its co-localization within a genomic region controlling variation both in aliphatic glucosinolate content (metabolite QTL) and in transcript level for genes involved in the biosynthesis of aliphatic glucosinolates (expression QTL), as well as its co-expression with genes in aliphatic glucosinolate biosynthesis. A phylogenetic analysis with the R2R3 motif of MYB28 showed that it and two homologues, MYB29 and MYB76, were members of an Arabidopsis-specific clade that included three characterized regulators of indole glucosinolates. Over-expression of the individual MYB genes showed that they all had the capacity to increase the production of aliphatic glucosinolates in leaves and seeds and induce gene expression of aliphatic biosynthetic genes within leaves. Analysis of leaves and seeds of single knockout mutants showed that mutants of MYB29 and MYB76 have reductions in only short-chained aliphatic glucosinolates whereas a mutant in MYB28 has reductions in both short- and long-chained aliphatic glucosinolates. Furthermore, analysis of a double knockout in MYB28 and MYB29 identified an emergent property of the system since the absence of aliphatic glucosinolates in these plants could not be predicted by the chemotype of the single knockouts. CONCLUSIONS/SIGNIFICANCE: It seems that these cruciferous-specific MYB regulatory genes have evolved both overlapping and specific regulatory capacities. This provides a unique system within which to study the evolution of MYB regulatory factors and their downstream targets.